Chronic inflammation plays a significant role in tumor promotion, migration and invasion. Using microarray analysis, we observed a profound increase in genes involved in pro-inflammatory pathways in epidermal growth factor receptor inhibitor (EGFRI)-treated head and neck squamous cell carcinoma (HNSCC) cell lines compared to their respective vehicle-treated cell lines. We hypothesized that the efficacy of EGFRIs may be offset by the pro-inflammatory response that these drugs produce in HNSCC tumor cells. We found that clinical EGFRIs such as erlotinib, cetuximab, lapatinib and panitumumab induced the secretion of pro-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-8, GM-CSF, TNFα and IFNγ. Focusing on IL-6, we found that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression and exogenous IL-6 was able to protect HNSCC cells from erlotinib-induced cytotoxicity. Conversely, an IL-6 receptor antagonist tocilizumab, sensitized HNSCC cells to erlotinib in vitro and in vivo. Inhibitors of NFκB, p38 and JNK suppressed erlotinib-induced IL-6 expression, suggesting an important role of NFκB and MAPK pathways in IL-6 expression. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced pro-inflammatory cytokines expression. Taken together, these results suggest that clinical EGFRIs induce the expression of pro-inflammatory cytokines via NOX4. Therefore, the anti-tumor activity of EGFRIs may be partially reduced by activation of NOX4-mediated pro-inflammatory pathways in HNSCC.

BackgroundDespite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab.MethodsParental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS).ResultsCetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels.ConclusionsAltogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0550-z) contains supplementary material, which is available to authorized users.

Epidermal growth factor receptor (EGFR) is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). However many HNSCC patients respond poorly to the EGFR inhibitors (EGFRIs) cetuximab and erlotinib despite tumor expression of EGFR. Gene expression analysis of erlotinib-treated HNSCC cells revealed an upregulation of genes involved in MyD88-dependent signaling compared to their respective vehicle-treated cell lines. We therefore investigated if MyD88-dependent signaling may reduce the anti-tumor efficacy of EGFRIs in HNSCC. Erlotinib significantly upregulated interleukin-6 (IL-6) secretion in HNSCC cell lines which our laboratory previously reported to result in reduced drug efficacy. Suppression of MyD88 expression blocked erlotinib-induced IL-6 secretion in vitro and increased the anti-tumor activity of erlotinib in vivo. There was little evidence of toll-like receptor or interleukin-18 receptor involvement in erlotinib-induced IL-6 secretion. However, suppression of interleukin-1 receptor (IL-1R) signaling significantly reduced erlotinib-induced IL-6 production. A time-dependent increase of IL-1 alpha (IL-1α) but not IL-1 beta (IL-1β) was observed in response to erlotinib treatment and IL-1α blockade significantly increased the anti-tumor activity of erlotinib and cetuximab in vivo. A pan-caspase inhibitor reduced erlotinib-induced IL-1α secretion suggesting that IL-1α was released due to cell death. Human HNSCC tumors showed higher IL-1α mRNA levels compared to matched normal tissue, and IL-1α was found to be negatively correlated with survival in HNSCC patients. Overall, the IL-1α/IL-1R/MYD88/IL-6 pathway may be responsible for the reduced anti-tumor efficacy of erlotinib and other EGFRIs; and blockade of IL-1 signaling may improve the efficacy of EGFRIs in the treatment of HNSCC.

Current therapies for gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that direct in vivo augmentation of the induction of gut-homing regulatory T (Treg) cells is a strategy of expected specificity for the treatment of chronic intestinal inflammation (e.g., inflammatory bowel disease). We showed that dendritic cells (DCs), engineered to de novo produce high concentrations of both 1,25-dihydroxyvitamin D, the active vitamin D metabolite, and retinoic acid, an active vitamin A metabolite, augmented the induction of T cells that express both the regulatory molecule Foxp3 and the gut-homing receptor CCR9 in vitro and in vivo. In vivo, the newly generated Ag-specific Foxp3+ T cells homed to intestines. Additionally, transfer of such engineered DCs robustly suppressed ongoing experimental colitis. Moreover, CD4+ T cells from spleens of the mice transferred with the engineered DCs suppressed experimental colitis in syngeneic hosts. The data suggest that the engineered DCs enhance regulatory function in CD4+ T cell population in peripheral lymphoid tissues. Finally, we showed that colitis suppression following in vivo transfer of the engineered DCs was significantly reduced when Foxp3+ Treg cells were depleted. The data indicate that maximal colitis suppression mediated by the engineered DCs requires Treg cells. Collectively, our data support that DCs de novo overproducing both 1,25-dihydroxyvitamin D and retinoic acid are a promising novel therapy for chronic intestinal inflammation.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) including erlotinib have demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date compared to the EGFR monoclonal antibody cetuximab. Therefore we seek to identify molecular mechanisms involved in tumor response to erlotinib and ultimately improve the anti-tumor efficacy of erlotinib for HNSCC patients. We have shown that erlotinib-treated HNSCC cell lines induced the expression and secretion of a wide variety of genes involved in inflammation and immune response including interleukin-1 (IL-1) and interleukin-6 (IL-6) compared to control-treated cells. The secretion of IL-6 from HNSCC cells in response to erlotinib was found to be triggered by MyD88-dependent IL-1 signaling and suppression of IL-1 signaling (using a neutralizing IL-1α antibody) significantly enhanced the anti-tumor efficacy of erlotinib in vivo. In erlotinib-resistant HNSCC cell lines, IL-1 signaling was significantly upregulated compared to their respective parental cell lines. Blockade of IL-1 signaling using a neutralizing IL-1α antibody or an IL-1 receptor (IL-1R) antagonist was able to significantly suppress the growth of erlotinib-resistant HNSCC xenografts as a single agent and in combination with erlotinib which was associated with reduced immune cell recruitment and angiogenesis. Altogether, blockade of the IL-1 pathway may represent a novel strategy to improve tumor response to erlotinib and other EGFR TKIs in HNSCC patients. Citation Format: Andrean Simons, Aditya Stanam, Adam Koch, Madelyn Espinosa-Cotton, Katherine Gibson-Corley. Targeting IL-1 signaling to improve tumor response to erlotinib in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 48.